Cancer cell enrichment system

ABSTRACT

Embodiments of the invention relate to a cell culture incubator having a gas flow regulation system that exerts control over atmospheric parameters within the incubator. Particular embodiments include an enclosed environmental chamber and a control unit operably linked thereto, the control unit having an oxygen module and a pressure module. Control unit embodiments, by way of these modules, are configured to regulate both oxygen partial pressure and total gas pressure within the enclosed environmental chamber. Embodiments of the control unit are adapted (a) to provide instructions to the oxygen module to regulate an oxygen level to an instructed hypoxic oxygen level and (b) to provide instructions to the pressure module to regulate total gas pressure to an instructed positive pressure level. The regulation of oxygen to the instructed hypoxic level prevails despite an oxygen partial pressure-increasing effect of the positive pressure condition associated with the instructed positive pressure level.

CROSS REFERENCE

This Application is a continuation-in-part of U.S. application Ser. No.15/566,337, filed Oct. 13, 2017, which is a national stage entry ofPCT/US2016/027881, filed Apr. 15, 2016, which claims the benefit of U.S.Provisional Application No. 62/149,268, filed Apr. 17, 2015, each ofwhich is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to instruments and systems for cell culture. Moreparticularly, the invention relates to cell culture systems that canregulate aspects of the atmospheric environment within a cell cultureincubator chamber.

BACKGROUND

Cell enrichment systems can be used to enrich, isolate, and expanddifferent populations of cells. These cell populations can include, forexample, cancer cells, circulating tumor cells, stem cells, and immunecells. Isolation and characterization of different cell types that areinduced through a cell enrichment system can be used to understand tumoretiology, the biology of metastasis, stem cell differentiation, immunecell proliferation, and to provide a biomarker for tumor progression.

Atmospheric conditions, such as the atmospheric pressure and theconcentrations of particular gases, such as oxygen, can be significantfactors in cell culture that can affect the growth rate, viability, andexpression of phenotypic aspects of various cell populations. Tounderstand these biological effects, cell culture instruments that canregulate various atmospheric conditions precisely and independently ofeach other could be valuable for research, diagnostic, and therapeuticgoals.

INCORPORATION BY REFERENCE

Each patent, publication, and non-patent literature cited in theapplication is hereby incorporated by reference in its entirety as ifeach was incorporated by reference individually.

SUMMARY OF THE INVENTION

Embodiments of the invention relate to a cell culture incubator thatincludes gas flow regulation system that exerts control over theatmospheric parameters to which cells in culture are exposed. Particularembodiments of the invention include an enclosed environmental chamberand a control unit operably linked to the enclosed environmentalchamber, the control unit having an oxygen module and a pressure module.Embodiments of the control unit, by way of these modules, are configuredto regulate both oxygen level and total gas pressure within the enclosedenvironmental chamber. Embodiments of the control unit are adapted (a)to provide instructions to the oxygen module to regulate an oxygen levelto an instructed hypoxic oxygen level and (b) to provide instructions tothe pressure module to regulate total gas pressure to an instructedpositive pressure level. Embodiments may also be instructed to operateat an ambient oxygen level and an ambient pressure level. The oxygenlevel and the total gas pressure level are regulated independently ofeach other. The regulation of oxygen to the instructed hypoxic levelprevails despite the oxygen partial pressure-increasing effect of thepositive pressure condition associated with the instructed positivepressure level.

Some embodiments of the cell culture incubator further include one ormore oxygen sensors configured to measure the oxygen level within theenclosed environmental chamber and to convey an informative signal tothe oxygen module, and one or more pressure sensors configured tomeasure the total atmospheric gas pressure within the enclosedenvironmental chamber and to convey an informative signal to thepressure module. Both the oxygen module and the pressure module arewithin the control unit. The control unit may include further controlunits related to the regulation of atmospheric parameters. Typically,atmospheric control units are configured to receive sensory input fromthe enclosed environmental chamber or the ambient environment, and todirect instructions to other elements of a gas flow regulation system.

Embodiments of a cell culture incubator and an included gas flowregulation system may include a nitrogen source operably connected tothe cell culture incubator, wherein the flow of nitrogen is regulated bythe control unit.

In some of these gas flow regulation embodiments, the regulated nitrogenflow is directed into the enclosed environmental chamber by way of achamber gas flow path, wherein the regulated nitrogen flow includes aresponse to oxygen sensor data by way of the oxygen module, and whereinthe response to a sensed oxygen level that is above the instructedoxygen level includes an instruction to flow nitrogen into theenvironmental chamber. In such embodiments and consequent response, as aresult of a dilution of oxygen within the enclosed environmental chamberby the inflow of nitrogen, the sensed oxygen level may come intocompliance with the instructed oxygen level. At that point, the controlunit may then instruct a cessation of the nitrogen flow into theenvironmental chamber.

In some of these gas flow regulation system embodiments for a cellculture incubator, the regulated nitrogen flow is directed into theenclosed environmental chamber by way of a chamber gas flow path, theregulated nitrogen flow includes a response to pressure sensor data byway of the pressure module, wherein the response to a pressure levelthat is below the instructed pressure level includes an instruction toflow nitrogen into the environmental chamber. In such embodiments andconsequent response, as a result of an increase in pressure level withinthe enclosed environmental chamber brought about by the inflow ofnitrogen, the pressure level may come into compliance with theinstructed pressure level. At that point, the control unit may theninstruct a cessation of the nitrogen flow into the environmentalchamber.

In some of these gas flow regulation system embodiments for a cellculture incubator, the regulation of pressure within the enclosedenvironmental chamber by the control unit includes a response to thepressure sensor, as mediated by the pressure module. In some of theseparticular embodiments, the regulation of pressure within the enclosedenvironmental chamber includes a response to a pressure lower than theinstructed pressure level, wherein such response to the high pressureincludes an inflow of nitrogen. In some embodiments, the regulation ofpressure within the enclosed environmental chamber includes a responseto a pressure lower than the instructed pressure level, wherein suchresponse to the high pressure includes an inflow of carbon dioxide. Insome embodiments, the regulation of pressure within the enclosedenvironmental chamber includes a response to a pressure lower than theinstructed pressure level, wherein such response to the low pressureincludes a cessation of inflow of carbon dioxide or a cessation ofinflow of nitrogen.

Some of these gas flow regulation system embodiments for a cell cultureincubator include regulation of the level of carbon dioxide, at least inpart, to engage a pH buffering system within the cell culture medium. Insome of these embodiments, the regulation of carbon dioxide flow intothe enclosed environmental chamber by the control unit include aresponse to the carbon dioxide sensor, as mediated by the carbon dioxidemodule. In some of these embodiments, the regulation of carbon dioxideflow into the enclosed environmental chamber by the control unitincludes a response to the pressure sensor, as mediated by the pressuremodule.

Particular embodiments of these gas flow regulation system embodimentsfor a cell culture incubator are directed toward regulating oxygen levelwithin the incubator to a hypoxic level. Accordingly, the oxygen levelwithin the enclosed environmental chamber is regulated by the oxygenmodule, the oxygen module providing instructions to regulate any one orboth of a flow of nitrogen or a flow of carbon dioxide into the enclosedenvironmental chamber. Inasmuch as oxygen level is typically regulatedto a level lower than that of the ambient level, approaches to loweringoxygen include dilution by addition of nitrogen or carbon dioxide, witha venting in order to keep total gas pressure at an instructed level. Inthe event that the oxygen level drifts below the instructed level, inputof air by an air injection pump provides an oxygen source.

In some embodiments of a gas flow regulation system, the instructions toregulate to an instructed hypoxic level include instructions to adjustthe oxygen level to a value within a range of about 0.1% to about 21%oxygen. In other embodiments, the instructions to regulate to aninstructed hypoxic level include instructions to adjust the oxygen levelto a value within a range of about 1.0% to about 12% oxygen. In otherembodiments, the instructions to regulate to an instructed hypoxic levelinclude instructions to adjust the oxygen level to a value within arange of about 2% to about 6% oxygen.

Particular embodiments of these gas flow regulation system embodimentsfor a cell culture incubator are directed toward regulating the totalgas pressure within the incubator to a level that is greater than theambient total gas pressure. The total gas pressure units used here(PSIG) refer to an amount of pressure over the ambient atmosphericpressure. In typical embodiments of the gas flow regulation system, thepressure level within the enclosed environmental chamber is regulated bythe pressure module, the pressure module providing instructions toregulate any one or more of a flow of nitrogen or a flow of carbondioxide, the inflow of either gas resulting in an increased pressure.

In some embodiments of the gas flow regulation system that are directedto creating a high pressure condition, the instructions to regulatepressure to an instructed positive pressure level include instructionsto adjust the pressure to a value within a range of about 0.5 PSIG toabout 30 PSIG. In some embodiments, the instructions to regulatepressure to an instructed positive pressure level include instructionsto adjust the pressure to a value within a range of about 1.0 PSIG toabout 20 PSIG. In some embodiments, the instructions to regulatepressure to an instructed positive pressure level include instructionsto adjust the pressure to a value within a range of about 2.0 PSIG toabout 10 PSIG. In some embodiments, the instructions to regulatepressure to an instructed positive pressure level include instructionsto adjust the pressure to a value within a range of about 2.5 PSIG toabout 5.0 PSIG.

In some embodiments, the invention provides a cell culture incubator,wherein the cell culture incubator comprises: a) an enclosedenvironmental chamber; and b) a control unit, wherein the control unitis operably linked to the enclosed environmental chamber, wherein thecontrol unit comprises a computer program product comprising acomputer-readable medium having computer-executable code encodedtherein, the computer-executable code adapted to encode: (i) an oxygenlevel module, wherein the oxygen level module is encoded to regulate anoxygen level of the enclosed environmental chamber, wherein the oxygenlevel module is encoded to control the removal of oxygen in the enclosedenvironmental chamber to generate a hypoxic oxygen level within theenclosed environmental chamber; (ii) a pressure module, wherein thepressure module is encoded to regulate the pressure of the enclosedenvironmental chamber, wherein the pressure module controls the additionof gas to generate a positive pressure condition in the enclosedenvironmental chamber; (iii) a temperature module, wherein thetemperature module is encoded to regulate the temperature of theenclosed environmental chamber; and (iv) a humidity module, wherein thehumidity module is encoded to regulate the humidity of the enclosedenvironmental chamber, wherein each of the oxygen level, pressure,temperature, and humidity mimics an in vivo microenvironment for a cell,wherein the cell culture incubator reaches each of an instructed oxygenlevel, pressure, temperature, and humidity within about 20 minutes ofreceiving an input of each of the instructed oxygen level, pressure,temperature, and humidity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an immunofluorescence image of a representative CTC cluster.

FIG. 2 is an illustrative workflow for enrichment of a targetsubpopulation of cells.

FIG. 3 depicts enrichment and propagation of a target subpopulation ofcells using a method of the invention.

FIG. 4 is an illustrative computer system to be used with a method ofthe invention.

FIG. 5 depicts results of biomarker assessment in CTCs from prostatecancer cells.

FIG. 6 shows results of biomarker determination for prostate cancercells.

FIG. 7 displays results of mRNA sequencing analysis for the nerve growthfactor signaling pathway.

FIG. 8 displays results of mRNA sequencing analysis for the Aurora Asignaling pathway.

FIG. 9 displays results of mRNA sequencing analysis for the Kit receptorsignaling pathway.

FIG. 10 is an immunofluorescence image for EPCAM expression in a PDACCTC.

FIG. 11 depicts results of NANOG signaling pathway expression in PDACversus mCRPC CTCs.

FIG. 12 depicts results of Wnt signaling pathway expression in PDACversus mCRPC CTCs.

FIG. 13 displays results of SNP and INDEL analysis for CTCs.

FIG. 14 displays results of SNP and INDEL analysis for CTCs.

FIG. 15 depicts an illustrative user interface for a system of theinvention.

FIG. 16 depicts an illustrative user interface for a system of theinvention.

FIG. 17 depicts an illustrative user interface for a system of theinvention.

FIG. 18 depicts an illustrative user interface for a system of theinvention.

FIG. 19 displays an illustrative cell culture incubator of theinvention.

FIG. 20 displays a door configuration of a cell culture incubator of theinvention.

FIG. 21 displays a door configuration of a cell culture incubator of theinvention.

FIG. 22 depicts a door heater of a cell culture incubator of theinvention.

FIG. 23 depicts increased cellular transfection efficiency using greenfluorescent protein.

FIG. 24A is an illustrative embodiment of gas flow control system to beused with a cell culture incubator and a method of the invention.

FIG. 24B is an illustrative embodiment of nitrogen flow system to beused with a cell culture incubator and a method of the invention.

DETAILED DESCRIPTION Method of the Invention

A method of the present invention can be used to isolate CTCs, and othertarget cell subpopulations, from a biological sample. A method of theinvention can be used, for example, to isolate cell populations,selectively separate cell populations, maintain cells in adifferentiated or an undifferentiated state, forcibly differentiatecells, enrich cell populations, expand cell populations (throughproliferation or selective enrichment), modulate functions of cellpopulations, modulate morphology of cell populations, modulateepigenetic characteristics, and modulate gene and protein expressionprofiles. A method of the invention can be used in, for example, primarycells, cell lines, or microbial communities.

Target cell subpopulations can include, for example, CTCs, cancer stemcells (CSCs), hematopoietic stem cells (HSCs), endothelial progenitorcells (EPCs), pre-cancerous cells, stem cells, fetal stem cells,undifferentiated stem cells, fetal cells, bone marrow cells, progenitorcells, foam cells, mesenchymal cells, epithelial cells, epithelialprogenitor cells, endothelial cells, endometrial cells, trophoblasts,cancer cells, red blood cells, white blood cells, immune system cells,connective tissue cells, hepatocytes, neurons, induced pluripotent stem(IPS) cells, or any combination thereof.

A method of the invention can be used, for example, to maintain neuronalcells in culture, to maintain hepatocytes in culture, and toxicityscreening. The invention can be used to differentiate IPS cells andstems cell into, for example, cells of the mesoderm, ectoderm, andendoderm. A method of the invention can be used to differentiate cellsinto neurons, cardiomyocytes, hepatocytes, hematopoietic stem cells,osteoblasts, osteoclasts, epithelial cells, endothelial cells,astrocytes, adipocytes, immune cells, mast cells, erythrocytes, oocytes,or spermatocytes.

CTCs can be composed of heterogeneous clusters of cancer and immunecells in vivo and can display differential expression ofimmunomodulatory and stem cell signaling pathway in vitro.

FIG. 1 displays immunofluorescence (IF) for a circulating tumor cellcluster from a subject with castration-resistant prostate cancer (CRPC).The IF image demonstrates that CTCs can contain both cancer and immunecells. The DAPI staining (indicated by oval-shaped cell staining)indicates the nuclei of the cells. A white blood cell (WBC) antibodycocktail can be used to detect, for example, CD3, CD14, CD16, CD19,CD20, CD45, and CD56, in the CTC cluster (white arrows in FIG. 1). Acytokeratin antibody cocktail can be used to detect, for example, CK4,CK5, CK6, CK8, CK10, CK13, and CK18 in the CTC cluster (fibrillarstaining seen in and around oval-shaped nuclei in FIG. 1). The brightwhite staining, and the fibrillar staining around the oval-shapednuclei, indicates prostate-specific membrane antigen (PSMA) andprostate-specific antigen (PSA) proteins. The numbers in the legendindicate the wavelength (nm) used for excitation of the stains.

FIG. 2 depicts an illustrative workflow that can be used to obtain anenriched population of cells from a heterogeneous cell population. Thesources of cells used in a method of the invention can include, forexample, fresh blood, white blood cells (WBC), the buffy layer ofcentrifuged blood, cryopreserved tumors and biopsies, samples forleukapheresis, fine needle aspirates, fresh biopsies, urine, or fecalmatter. After obtaining a sample from a subject, the sample can beprepared for use in a cell isolation kit to separate, for example, ablood sample into plasma, white blood cells and platelets, and red bloodcells. CTCs can be found in the white blood cell and platelet fractionof centrifuged blood. The cell isolation step can be about 30 minuteslong. After the heterogeneous cell population has been isolated, thecells can be applied to the enrichment medium for propagation andenrichment of viable CTC colonies. The adhesion of the cells to thesubstrate can take from a few hours to about one day. Proliferationduring culture of the cells can be observed within about 1 to 3 daysafter adherence, after which next-generation sequencing (NGS) can beperformed on the cell colonies for the markers of interest.Next-generation sequencing methods can include, for example, wholegenome sequencing, whole genome resequencing, whole exome sequencing,whole transcriptome mRNA sequencing, ChIP-sequencing, andbioinformatics.

FIG. 3 depicts adherence and propagation of a collected heterogeneouscell population from a subject. First, the cells can be applied to aplate, which can be coated with a substance, such as a growthfactor-infused hydrogel. After about 30 minutes, the cells are adheredto the plate. Over the next two hours, the cells spread across theplate, and the growth medium is exchanged allowing for removal of anyremaining white bloods cells (WBCs). The media is replaced with achemically-defined culture medium to promote the growth of CTC colonies.The cells can be grown in an environment that can be adjusted to mimic,for example, the tumor microenvironment, via changes in oxygen orpressure levels to obtain viable CTCs. In some embodiments, the cellsare grown in hypoxic conditions.

The present invention can use a substrate to capture target cellsubpopulations from a sample. The heterogeneous cell population can beapplied to, for example, a culture dish coated with a substrate that canpromote growth and enrichment of the target cell population. The targetcell subpopulation can adhere to the substrate with higher affinity thanother cells, for example, white blood cells. Cells that do not adhere tothe substrate can be washed away with media or maintained in culture.Once adhered, the cells can spread and begin dividing on the substrate.

The substrate can comprise, for example, 1, 2, 3, 4, or 5 layers. Thedistance between two substrates layers may range from about 0.001 toabout 20 mm, about 1 to about 10 mm, or about 1 to about 5 mm and eachlayer can be about 0.001, about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 12, about 15, about17, or about 20 mm.

The cells can be plated on a material made of, for example, plastic,glass, gelatin, polyacrylamide, or any combination thereof. The dishesused to the plate the cells can be, for example, microscope slides,culture plates, culture dishes, Petri dishes, microscope coverslips, anenclosed environmental chamber, a sealed culture dish, or multi-wellculture dishes.

The binding surface layer of the substrate can be the portion of thesubstrate that is in contact with the captured cells. In some instances,the binding surface layer is the only layer, adjacent to the base layer,or separated from the base layer by one or more middle layers.

The binding surface layer of the substrate can comprise, for example,cell monolayers, cell lysates, biological materials associated with theextracellular matrix (ECM), gelatin, or any combination thereof.

Biological materials associated with the ECM can include, for example,collagen type I, collagen type IV, laminin, fibronectin, elastin,reticulin, vimentin, hygroscopic molecules, glycosaminoglycanse,proteoglycans, roteoglycans, glycocalyx, bovine serum albumin, humanserum albumin, Poly-L-lysine, Poly-D-lysine, or Poly-L-ornithine. Thegelatin can be from an animal source, for example, the gelatin canporcine or bovine.

The monolayer of cells used in the substrate can be, for example,mammalian cells, endothelial cells, vascular cells, venous cells,capillary cells, human umbilical vein endothelial cells (HUVEC), humanlung microvascular endothelial cells (HLMVEC), human keratinocytes,human mesenchymal stem cells, human bone marrow stromal cells, and humanastroglial cells. The cell lines can be obtained from a primary sourceor from an immortalized cell line. The monolayer of cells can beirradiated by ultraviolet light or X-ray sources to cause senescence ofcells. The monolayer can also contain a mixture of one or more differentcell types. The different cell types may be co-cultured together. Onenon-limiting example of co-culture is a combination of primary humanendothelial cells co-cultured with transgenic mouse embryonicfibroblasts mixed to form a monolayer.

The binding surface layer of the substrate can contain, for example, amixture of intracellular components. One method that can be used toobtain a mixture of intracellular components is lysis of the cells andcollection of the cytosolic and cytoskeletal components. The lysed cellsmay be primary or immortalized. The lysed cells can be from either mono-or co-cultures.

The binding surface layer of the substrate can contain biologicalmaterials associated with the extracellular matrix (ECM) or bindingmoieties such as hyaluronic acid hydrogels. For example, gelatin can bemixed directly with cells, binding moieties, biological materialsassociated with the ECM, or any combination thereof, to make a bindingsurface layer for the substrate. For example, the binding surface layercan comprise a gelatin mixed with a collagen.

The substrate can have one or more middle layers. The middle layer ofthe substrate can be one or more monolayers of cells. The cells of themonolayer can be of varying origin. For example, the middle layer of thesubstrate can be made by growing a confluent monolayer of mouseembryonic fibroblasts on the base layer and then growing another layerof cells, for example, the binding surface layer, on top of theconfluent mouse embryonic fibroblasts.

A feeder layer can be used in the substrate for growth and enrichment ofthe target cell subpopulation. A feeder layer can sit adjacent to a baselayer and can be separated from the binding surface layer of thesubstrate. The feeder layer can be a monolayer of feeder cells. Thecells of the monolayer can be of varying origin. For example, the feederlayer can be made by growing a monolayer of human endothelial cells ormouse embryonic fibroblasts on a base layer.

Conjugation of layers of the substrate can be done by allowing cells togrow in a monolayer on top of the base layer or middle layer.Conjugation of layers can also be done by pre-treating the surface witha surface of either net positive, net negative, or net neutral charge.The conjugation procedure can be aided by chemical moieties, linkers,protein fragments, nucleotide fragments, or any combination thereof.

The configuration and composition of the substrate can be tailored forenrichment of a particular target cell subpopulation. The composition ofthe substrate can vary based on, for example, patient type, cancer type,stage of cancer, patient medical history, and genomic and proteomicanalysis of the patient tumor.

The enrichment media used for growing the cells can be supplemented ormade with culture media that has been collected from cell cultures,blood plasma, or any combination thereof. The enrichment media can be,for example, Plating Culture Medium, Type R Long Term Growth Medium,Type DF Long Term Growth Medium, Type D Long Term Growth Medium, andMEF—Enrichment Medium, or any combination thereof. The enrichment mediumcan contain, for example, a primary nutrient source, animal serum, ions,elements, calcium, glutamate, magnesium, zinc, iron, potassium, sodium,amino acids, vitamins, glucose, growth factors, hormones, tissueextracts, proteins, small molecules, or any combination thereof.

Non-limiting examples of amino acids that can used in the enrichmentmedia include essential amino acids, phenylalanine, valine, threonine,tryptophan, isoleucine, methionine, leucine, lysine, and histidine,arginine, cysteine, glycine, glutamine, proline, serine, tyrosine,alanine, asparagine, aspartic acid, glutamic acid, or any combinationthereof.

Non-limiting examples of growth factors that can be used in theenrichment media include epidermal growth factor (EGF), nerve growthfactor (NGF), brain derived neurotrophic factor (BDNF), fibroblastgrowth factor (FGF), stem cell factor (SCF), insulin-like growth factor(IGF), transforming growth factor-beta (TGF-β), basic fibroblast growthfactor (bFGF), testosterone, estrogen, thyroid-stimulating hormone(TSH), follicle-stimulating hormone, luteinizing hormone, eicosanoids,melatonin, thyroxine, vasopressin, oxytocin, or any combination thereof.

Non-limiting examples of hormones include peptide hormones, insulin,steroidal hormones, hydrocortisone, progesterone, testosterone,estrogen, dihydrotestosterone, or any combination thereof.

Non-limiting examples of tissue extracts include pituitary extract.Non-limiting examples of small molecule additives include sodiumpyruvate, endothelin-1, transferrin, cholesterol, or any combinationthereof.

Non-limiting examples of other components that can be used in theenrichment media include pipecolic acid, gamma-Aminobutyric acid (GABA),human serum albumin, bovine serum albumin, glutathione, humanalpha-fetoprotein, bovine alpha-fetoprotein, human holo-transferrin, orany combination thereof.

Non-limiting examples of salts that can be used in the enrichment mediainclude calcium chloride, magnesium chloride, sodium bicarbonate,magnesium sulfate, sodium chloride, citrate, potassium phosphate, sodiumphosphate, or any combination thereof.

In some embodiments, the enrichment media contains pipecolic acid, GABA,bFGF, TGFβ-1, human insulin, human holo-transferrin, human serumalbumin, and reduced glutathione.

The amino acids, growth factors, hormones, tissue extracts, salts, orany other component that can be used in the enrichment media can be at aconcentration of, for example, about 0.001 nM, about 0.005 nM, about0.01 nM, about 0.015 nM, about 0.02 nM, about 0.25 nM, about 0.03 nM,about 0.035 nM, about 0.04 nM, about 0.045 nM, about 0.05 nM, about0.055 nM, about 0.06 nM, about 0.065 nM, about 0.07 nM, about 0.075 nM,about 0.08 nM, about 0.085 nM, about 0.09 nM, about 0.1 nM, about 0.015nM, about 0.2 nM, about 0.25 nM, about 0.3 nM, about 0.35 nM, about 0.4nM, about 0.45 nM, about 0.5 nM, about 0.55 nM, about 0.6 nM, about 0.65nM, about 0.7 nM, about 0.75 nM, about 0.8 nM, about 0.85 nM, about 0.9nM, about 0.95 nM, about 0.001 μM, about 0.005 μM, about 0.01 μM, about0.015 μM, about 0.02 μM, about 0.025 μM, about 0.03 μM, about 0.035 μM,about 0.04 μM, about 0.045 μM, about 0.05 μM, about 0.055 μM, about 0.06μM, about 0.065 μM, about 0.07 μM, about 0.075 μM, about 0.08 μM, about0.085 μM, about 0.09 μM, about 0.085 μM, about 0.09 μM, about 0.085 μM,about 0.09 μM, about 0.085 μM, about 0.09 μM, about 0.085 μM, about 0.09μM, about 0.085 μM, about 0.09 μM, about 0.085 μM, about 0.09 μM, about0.095 μM, about 0.1 μM, about 0.15 μM, about 0.2 μM, about 0.25 μM,about 0.3 μM, about 0.35 μM, about 0.4 μM, about 0.45 μM, about 0.5 μM,about 0.55 μM, about 0.6 μM, about 0.65 μM, about 0.7 μM, about 0.75 μM,about 0.8 μM, about 0.85 μM, about 0.9 μM, about 0.95 μM, about 0.001mM, about 0.005 nM, about 0.01 mM, about 0.015 mM, about 0.02 mM, about0.025 mM, about 0.03 mM, about 0.035 mM, about 0.04 mM, about 0.045 mM,about 0.05 mM, about 0.055 mM, about 0.06 mM, about 0.065 mM, about 0.07mM, about 0.075 mM, about 0.08 mM, about 0.085 mM, about 0.09 mM, about0.095 mM, about 0.1 mM, about 0.15 mM, about 0.2 mM, about 0.25 mM,about 0.3 mM, about 0.35 mM, about 0.4 mM, about 0.45 mM, about 0.5 mM,about 0.55 mM, about 0.6 mM, about 0.65 mM, about 0.7 mM, about 0.75 mM,about 0.8 mM, about 0.85 mM, about 0.9 mM, about 0.95 mM, about 1 mM,about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM,about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about500 mM, about 600 mM, about 700 mM, about 800 nM, about 900 mM, andabout 1 M.

The culturing conditions in a method of the invention can be adjusted tosimulate oxygen and pressure levels found in a particularmicroenvironment to promote the collection of a desired cell population.The microenvironment can be, for example, a tumor microenvironment, bonemetastatic environment, vasculature environment, or brainmicroenvironment. The oxygen level used during culturing conditions orin a cell incubator can be, for example, about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about20%, about 21%, about 22%, about 23%, about 24%, or about 25% oxygen inthe incubator. In some embodiments, the cells can be grown under hypoxicconditions.

The culturing condition in a method of the invention can be adjusted tosimulate the pressure found in the tumor microenvironment to promote thecollection of, for example, CTCs, maintenance of a tumor biopsy, orexpansion of a tumor biopsy. The pressure used during culturingconditions can be a PSI gauge (PSIG) reading of, for example, about 0.5PSIG, about 0.6 PSIG, about 0.7 PSIG, about 0.8 PSIG, about 0.9 PSIG,about 1 PSIG, about 1.1 PSIG, about 1.2 PSIG, about 1.3 PSIG, about 1.4PSIG, about 1.5 PSIG, about 1.6 PSIG, about 1.7 PSIG, about 1.8 PSIG,about 1.9 PSIG, about 2 PSIG, about 2.5 PSIG, about 3 PSIG, about 3.5PSIG, about 4 PSIG, about 4.5 PSIG, about 5 PSIG, about 6 PSIG, about 7PSIG, about 8 PSIG, about 9 PSIG, about 10 PSIG, about 15 PSIG, about 20PSIG, about 25 PSIG, about 30 PSIG, about 35 PSIG, about 40 PSIG, about45 PSIG, about 50 PSIG, or about 55 PSIG.

The pressure used during culturing conditions can be, for example, about3.45 kPa, about 4.14 kPa, about 4.83 kPa, about 5.52 kPa, about 6.21kPa, about 6.89 kPa, about 7.58 kPa, about 8.27 kPa, about 8.96 kPa,about 9.65 kPa, about 10.3 kPa, about 11 kPa, about 11.7 kPa, about 12.4kPa, about 13.1 kPa, about 13.8 kPa, about 17.2 kPa, about 20.7 kPa,about 24.1 kPa, about 27.6 kPa, about 31 kPa, about 34.4 kPa, about 41.4kPa, about 48.3 kPa, about 55.2 kPa, about 62.1 kPa, about 68.9 kPa,about 103 kPa, about 138 kPa, about 172 kPa, about 207 kPa, about 241kPa, about 276 kPa, about 310 kPa, about 345 kPa, or about 379 kPa.

The pH of the enrichment media used in a method of the invention can be,for example, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1,about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4,about 4.5, about 4.55, about 4.6, about 4.7, about 4.8, about 4.9, about5, about 5.5, about 6, about 6.5, about 6.6, about 6.7, about 6.8, about6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5,about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.5, about 9,about 9.5, about 10, about 10.5, or about 11 pH units.

The viscosity of the enrichment media can be adjusted by, for example,at least 0.001 Pascal-second (Pa·s), at least 0.001 Pa·s, at least0.0009 Pa·s, at least 0.0008 Pa·s, at least 0.0007 Pa·s, at least 0.0006Pa·s, at least 0.0005 Pa·s, at least 0.0004 Pa·s, at least 0.0003 Pa·s,at least 0.0002 Pa·s, at least 0.0001 Pa·s, at least 0.00005 Pa·s, or atleast 0.00001 Pa·s, depending on the cell types being cultured.

The oxygen solubility of the enrichment media can be, for example, about0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%,about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about96%, about 97%, about 98%, or about 99%.

A method of the invention can further comprise coating surfaces for celladhesion with particular media compositions to promote cellular andcellular protein binding to the surface. The surface can be, forexample, a cell culture plate, a cell culture plate with multiple wells,a petri dish, a glass slide, a cover slip, or a glass dish. The mediaused for coating of the cell adhesion surfaces can include, for example,3-(aminopropyl)-trimethoxysilane, (3-mercapto-propyl)trimethoxysilane,(3-Aminopropyl)triethoxysilane, N-[3-(trimethoxysilyl)-propyl]-ethylenediamine,(3-Glycidyloxypropyl)trimethoxysilane,[3-(2-aminoethyl-amino)-propyl]trimethoxysilane,trimethoxy[3-(methylamino)propyl]silane,3-aminopropyl(diethoxy)-methylsilane, or glutaraldehyde.

The surface coating can further comprise an extracellular matrix (ECM)mix to facilitate cell binding. The mix can include, for example,collagens, basement membrane proteins, collagen IV, laminins,fibronectin, vitronectin, vimentin, tumor-derived extracellular matrixproteins, or inert self-assembling peptides systems. The components usedcan be animal- or human-derived. The ECM mix can be diluted to a pH ofabout 4 to about 10 using, for example, potassium hydroxide (KOH),1-glycine, DMEM powder, sodium hydroxide (NaOH), or PBS. The ECM mix canbe further supplemented with, for example, human plasma oranimal-derived serum. The animal-derived serum can be, for example,bovine serum or fetal bovine serum.

The cells can be cultured in enrichment media, or in a cell cultureincubator, for about 1 minute, about 2 minutes, about 3 minutes, about 4minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20minutes, about 25 minutes, about 30 minutes, about 40 minutes, about 50minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours,about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours,about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 2days, about 3 days, about 4 days, about 5 days, about 6 days, about 1week, about 2 weeks, about 3 weeks, about 4 weeks, about 2 months, about3 months, about 4 months, about 5 months, about 6 months, about 7months, about 8 months, about 9 months, about 10 months, about 11months, about 1 year, about 1.5 years, about 2 years, about 2.5 years,or about 3 years.

Databases containing information regarding genetic mutations that areprevalent in specific types of cancer can be used to compare the geneticprofile or biomarker expression of the target subpopulations derivedusing the present invention to known mutations. Non-limiting examples ofdatabases that can be used for comparison include COSMIC, cBio Portal,Human Gene Mutation Database (HGMD TM), GWAS central, and the UniversalMutation Database.

Cell Culture Incubator

The invention further provides a cell culture incubator. The cellculture incubator can comprise an enclosed environmental chamber. Thecell culture incubator can be configured to maintain a gas compositionof the enclosed environmental chamber, an atmospheric pressure of theenclosed environmental chamber, humidity, carbon dioxide level, oxygenlevel, and an internal ambient temperature of the enclosed environmentalchamber. The cell culture incubator can comprise a control unit, whereinthe control unit can be configured to maintain at least one of the gascomposition, the atmospheric pressure, humidity, carbon dioxide level,oxygen level, or the internal ambient temperature. The control unit canbe operably linked to the enclosed environmental chamber. The controlunit can be configured to maintain at least two of the gas compositions,the atmospheric pressure, humidity, carbon dioxide level, oxygen level,and the internal ambient temperature. The control unit can be configuredto maintain the gas composition, the atmospheric pressure, humidity,carbon dioxide level, oxygen level, and the internal ambienttemperature. The control unit of the cell culture incubator can beuser-controlled or automated based on sensors in the cell cultureincubator. The control unit can be configured to create a dynamic gascomposition, atmospheric pressure, humidity, carbon dioxide level,oxygen level and the internal ambient temperature as a function of time.The control unit can be configured to cycle between several differentgas compositions, atmospheric pressure, humidity level, carbon dioxidelevel, oxygen level, and the internal ambient temperature as a functionof time, and can be stochastic or periodic. Some aspects of embodimentsof the cell culture incubator are described further below in the sectionentitled “Gas Flow Regulation System”, and are depicted in FIGS.24A-24B.

At least one of the gas composition, the atmospheric pressure, humidity,carbon dioxide level, oxygen level, and the internal ambient temperaturecan be configured for selective proliferation of a target primary cellsubpopulation as compared to a non-target primary cell subpopulation.The selective proliferation of a target cell subpopulation can beevidenced by, for example, a two-fold increase in the proliferation rateof the target primary cell subpopulation as compared to theproliferation rate of the non-target primary cell subpopulation. Atleast one of the gas composition, the atmospheric pressure, humidity,carbon dioxide level, oxygen level, and the internal ambient temperaturecan be configured for selective adherence of a target primary cellsubpopulation as compared to a non-target primary cell subpopulation.The selective adherence of a target primary cell subpopulation can beevidenced by a two-fold increase in adherence of the target primary cellsubpopulation as compared to adherence of the non-target primary cellsubpopulation. At least one of the gas composition, the atmosphericpressure, humidity, carbon dioxide level, oxygen level, and the internalambient temperature can be configured to promote selective colonyformation of the target primary cell as compared to colony formation ofthe non-target primary cell subpopulation. The selective colonyformation can be evidenced by a two-fold increase in colony formation ofthe target primary cell subpopulation as compared to colony formation ofthe non-target primary cell subpopulation. The colony formation can be atwo-dimensional or three-dimensional colony formation.

The gas composition in the cell culture incubate can comprise an oxygenlevel between about 0.1 to about 21%. In some embodiments, the cellculture incubator maintains an oxygen level of the enclosedenvironmental chamber of no more than about 5%. In some embodiments, thecell culture incubator maintains an oxygen level of the enclosedenvironmental chamber of no more than about 2%. In some embodiments, thecell culture incubator maintains an oxygen level of the enclosedenvironmental chamber of no more than about 1%.

The cell culture incubator can maintain a user-controlled or automatedatmospheric pressure of the enclosed environmental chamber of about 1PSIG (6.89 kPa) or greater. In some embodiments, the cell cultureincubator maintains a user-controlled atmospheric pressure of theenclosed environmental chamber of about 2 PSIG (13.8 kPa) or greater. Insome embodiments, the cell culture incubator maintains a user-controlledatmospheric pressure of the enclosed environmental chamber of about 5PSIG (34.5 kPa). The cell culture incubator can maintain the atmosphericpressure of the enclosed environmental chamber by controlling an inletgas pressure.

The cell culture incubator can comprise a user interface. The userinterface can be configured to allow a user to control the gascomposition, oxygen level, carbon dioxide level, humidity, atmosphericpressure, or internal ambient temperature. The user interface can beconfigured to provide a display of the gas composition to a user. Theuser interface can be configured to provide a display of the oxygenlevel, carbon dioxide level, humidity, atmospheric pressure of theenclosed environmental chamber, and internal ambient temperature to auser. The cell culture incubator can be configured to maintain aninternal humidity of the enclosed environmental chamber. The enclosedenvironmental chamber can comprise a shelf, a pressure sensor, an oxygensensor, a carbon dioxide sensor, a temperature sensor, or an oxygenremoval catalyst. The shelf in the enclosed environmental chamber can bemade of, for example, stainless steel, silver, gold, or copper. In someembodiments, the shelf of the enclosed environmental chamber is a coppershelf.

The cell culture incubator can be operably linked to a gas tank. The gastank can comprise a CO2 tank, a nitrogen gas tank, an oxygen gas tank, agas tank comprising a defined mixture of one or more gases, or anycombination thereof. The cell culture incubator can be operably linkedto the gas tank via a pressurized pump or pressure sensor (e.g., apressure gauge). The pressurized pump or pressure sensor can maintain acontrolled flow of gas from the gas tank to the enclosed environmentalchamber of the cell culture incubator. The controlled flow of gas fromthe one or more tanks can have a set inlet pressure, the set inletpressure of the one or more tanks configured to maintain the desiredinternal gas composition or internal atmospheric pressure of theenclosed environmental chamber. The enclosed environmental chamber canhave a vacuum seal on the door of the enclosed environmental chamber.The enclosed environment chamber can be sealed by an inflatable seal.

The incubator can comprise a pressurized door. In some embodiments, theincubator comprises an outer pressurized door and an inner pressurizeddoor. The outer pressurized door and/or inner pressurized door can be,e.g., a double-walled door. The double-walled door can have avacuum-sealed latch. The outer pressurized door may include anintegrated pressure sensor on the door. The incubator can comprise adoor entry. The door entry can provide an entrance into an enclosedenvironmental chamber. Dimensions of the door entry opening can be lessthan the pressurized door. The door entry can comprise a rubber gasket.The rubber gasket can create a pressurized seal.

The cell culture incubator can comprise an integrated pressure sensor.The integrated pressure sensor can be a manifold pressure sensor. Theintegrated pressure sensor can be a water-based or silicon basedpressure sensor. The incubator can comprise a sterilization unit. Thesterilization unit can be a UV-based sterilization unit. The UV-basedsterilization unit can be configured to provide UV rays to the entirespace of an enclosed environmental chamber of the incubator. Theincubator can comprise a CO2 sensor. The CO2 sensor can be configured toprovide a detectable alarm upon deviation of +/−0.5% from a defined CO2level of the enclosed environmental chamber. The incubator can comprisean enclosed environmental chamber. The incubator can comprise a waterhumidity tray. The water humidity tray can promote sterility of theenclosed environmental chamber. The water humidity tray may be tethereddirectly to humidity sensor or regulator. The incubator can comprise anair jacket. The air jacket can maintain optimal temperature and propergas regulation. The air jacket can be a physically distinct compartment.The air jacket may house electrical controllers and circuit boards. Theincubator can comprise an oxygen removal catalyst and sensor forregulating oxygen levels within the incubator.

The incubator can comprise a heating element or temperature control. Theheating element or temperature control can comprise silent fan-basedheating elements. The silent fan-based heating elements can dispense aconstant flow of heated air into the air-jacket compartment. Passiveheating can then warm the inner chamber in an evenly distributed andconstant manner. The incubator can comprise a pressurized pump andregulator configured to provide a defined gas composition and internalatmospheric pressure. A motor based pump can dispense defined gasmixtures to maintain chamber pressure and gas composition levels (e.g.,1% oxygen, 5% CO2, 94% N2). The incubator can comprise a user interface.A user can use the user interface to set gas levels and pressures. Theuser interface can be integrated to the sensor and pump for directcontrol. The pressurized pump and regulator can comprise a gas inlet.The gas inlet can allow flow of any gas into the enclosed environmentalchamber. For example, the gas inlet can be connected to an oxygen tank.The gas inlet can be connected to a CO2 tank. The gas inlet can beconnected to a nitrogen tank. In some embodiments, the incubatorcomprises a gas inlet connected to an oxygen tank, a gas inlet connectedto a CO2 tank, and a gas inlet connected to a nitrogen tank. The gasinlet can be connected to a tank containing a custom gas mixture. Insome embodiments, a user can control a flow of gas through any gasinlet. Any one of the gas inlets may be connected to a flow meter. Theflow meter can regulate an inlet gas pressure. The cell cultureincubator can comprise a humidity control unit/sensor. The humiditycontrol unit/sensor can be directly connected to the water humiditytray.

The enclosed environmental chamber of the cell culture incubator cancomprise a sterilization unit, which can be a UV sterilization unit. Theenclosed environmental chamber can comprise a pressurized door. Theenclosed environmental chamber can comprise a sensor that provides adetectable alarm upon detection of an oxygen level of the enclosedenvironmental chamber that differs by more than about ±0.5% from auser-desired oxygen level. The enclosed environmental chamber cancomprise a sensor that provides a detectable alarm upon detection of anatmospheric pressure of the enclosed environmental chamber that differsby more than about ±0.5% from a user-desired atmospheric pressure. Theenclosed environmental chamber can comprise a user display that displaysan atmospheric pressure level of the enclosed environmental chamber. Insome embodiments, the enclosed environmental chamber comprises a userdisplay that displays an O2 level of the enclosed environmental chamber.In some embodiments, the enclosed environmental chamber comprises a userdisplay which displays a CO2 level of the enclosed environmentalchamber. In some embodiments, the enclosed environmental chambercomprises a user display which displays a temperature level of theenclosed environmental chamber.

A user can program the cell culture incubator to mimic, for example,physiological, tumor microenvironment, hypoxic, high pressure, lowpressure, or supraphysiological conditions. The cell culture incubatorcan be configured to calibrate to the conditions set by the user withinabout one minute, about 2 minutes, about 3 minutes, about 4 minutes,about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes,about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes,about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes,about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes,about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes,about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes,about 45 minutes, about 50 minutes, about 55 minutes, or about one hour.In some embodiments, the cell culture incubator can reach the desiredconditions set by the user in less than about 20 minutes. In someembodiments, the cell culture incubator can reach the desired conditionsset by the user in about 20 minutes. In some embodiments, the cellculture incubator can reach the desired conditions set by the userwithin 20 minutes.

In some embodiments, the enclosed environmental chamber occupies no morethan 6 cubic feet of space. In some embodiments, the enclosedenvironmental chamber occupies no more than 3.5 cubic feet of space. Insome embodiments, the enclosed environmental chamber occupies no morethan 2 cubic feet of space. In some embodiments, the enclosedenvironmental chamber occupies no more than 1.5 cubic feet of space. Insome embodiments, the enclosed environmental chamber occupies no morethan 1 cubic foot of space. In some embodiments, the enclosedenvironmental chamber occupies less than 1 cubic foot of space. In someembodiments, the cell culture plate comprises 1, 6, 12, 24, 48, 96, 384,1056, 1536, or 3456 wells.

A method of the invention can employ a cell culture incubator forculturing of a target cell population. FIG. 19 provides an illustrativeexample of a cell culture incubator that can be used in a system of theinvention. 1901 is the door of the incubator that can be opened to placea cell culture in the incubator. 1902 is a control unit that can be usedto program the cell culture incubator using parameters including, forexample, temperature, humidity, oxygen level, carbon dioxide level, timeof incubation, nitrogen level, and chamber pressure. 1903 is a USB portthat can be used to input data to or extract data from the cell cultureincubator.

FIG. 20 is a diagram of the front of the cell culture incubator. FIG. 20depicts the door, a rubber gasket around the edge of the door that canfit tightly using a door handle latch, retaining lip, open and closebuttons for the door, touch screen, rotary actuator or motor, andchamber of the incubator. FIG. 21 shows the cell culture incubator doorclosed (2101) and open (2102). FIG. 22 depicts a door heater than can beused in a system of the invention. The figure shows the front plate,heating element, poron insulation, back plate, mica washers, copperplate, precision hollow shaft, and paddles that can be used to heat thecell culture incubator to a desired temperature.

The height, width, depth, or length of the cell culture incubator canbe, for example, about 6 in, about 6.5 in, about 7 in, about 7.5 in,about 8 in, about 8.5 in, about 9 in, about 9.5 in, about 10 in, about10.5 in, about 11 in, about 11.5 in, about 12 in, about 12.1 in, about12.2 in, about 12.3 in, about 12.4 in, about 12.5 in, about 12.6 in,about 12.7 in, about 12.8 in, about 12.9 in, about 13 in, about 13.1 in,about 13.2 in, about 13.3 in, about 13.4 in, about 13.5 in, about 13.6in, about 13.7 in, about 13.8 in, about 13.9 in, about 14 in, about 14.5in, about 15 in, about 15.5 in, about 16 in, about 16.5 in, about 17 in,about 17.5 in, about 18 in, about 18.5 in, about 19 in, about 19.5 in,about 20 in, about 20.5 in, about 21 in, about 21.5 in, about 22 in,about 22.5 in, about 23 in, about 23.5 in, about 24 in, about 24.5 in,about 25 in, about 25.5 in, about 26 in, about 26.5 in, about 27 in,about 27.5 in, about 28 in, about 28.5 in, about 29 in, about 29.5 in,about 30 in, about 30.5 in, about 31 in, about 31.5 in, about 32 in,about 32.5 in, about 33 in, about 33.5 in, about 34 in, about 34.5 in,about 35 in, about 35.5 in, about 36 in, about 36.5 in, about 37 in,about 37.5 in, about 38 in, about 38.5 in, about 39 in, about 39.5 in,about 40 in, about 40.5 in, about 41 in, about 41.5 in, about 42 in,about 42.5 in, about 43 in, about 43.5 in, about 44 in, about 44.5 in,about 45 in, about 45.5 in, about 46 in, about 46.5 in, about 47 in,about 47.5 in, about 48 in, about 48.5 in, about 49 in, about 49.5 in,about 50 in, about 50.5 in, about 51 in, about 51.5 in, about 52 in,about 52.5 in, about 53 in, about 53.5 in, about 54 in, about 54.5 in,about 55 in, about 55.5 in, about 56 in, about 56.5 in, about 57 in,about 57.5 in, about 58 in, about 58.5 in, about 59 in, about 59.5 in,about 60 in, or any combination thereof.

In some embodiments, the height of the cell culture incubator is 12 in.In some embodiments, the width of the cell culture incubator is 13.5 in.In some embodiments, the depth of the cell culture incubator is 13.1 in.

The capacity of the enclosed environmental chamber can be, for example,about 100 inch3, about 110 inch3, about 120 inch3, about 130 inch3,about 140 inch3, about 150 inch3, about 160 inch3, about 170 inch3,about 180 inch3, about 190 inch3, about 200 inch3, about 205 inch3,about 210 inch3, about 211 inch3, about 212 inch3, about 213 inch3,about 214 inch3, about 215 inch3, about 216 inch3, about 217 inch3,about 218 inch3, about 219 inch3, about 220 inch3, about 221 inch3,about 222 inch3, about 223 inch3, about 224 inch3, about 225 inch3,about 226 inch3, about 227 inch3, about 228 inch3, about 229 inch3,about 230 inch3, about 240 inch3, about 250 inch3, about 260 inch3,about 270 inch3, about 280 inch3, about 290 inch3, about 300 inch3,about 310 inch3, about 320 inch3, about 330 inch3, about 340 inch3,about 340 inch3, about 350 inch3, about 360 inch3, about 370 inch3,about 380 inch3, about 390 inch3, about 400 inch3, about 420 inch3,about 440 inch3, about 460 inch3, about 480 inch3, or about 500 inch3.In some embodiments, the capacity of the enclosed environmental chamberis about 220 inch3. In some embodiments, the capacity of the enclosedenvironmental chamber is about 221 inch3. In some embodiments, thecapacity of the enclosed environmental chamber is about 222 inch3. Insome embodiments, the capacity of the enclosed environmental chamber isabout 223 inch3. In some embodiments, the capacity of the enclosedenvironmental chamber is about 224 inch3. In some embodiments, thecapacity of the enclosed environmental chamber is 224 inch3.

Materials that can be used in the manufacture of the cell cultureincubator include, for example, stainless steel, glass, copper, silver,gold, plastic, blanket batting, hard-board insulation, or anycombination thereof. The enclosed environmental chamber can be made of,for example, copper or stainless steel. In some embodiments, theenclosed environmental chamber is made of copper.

The cell culture incubator can be maintained at a desired humiditylevel. The humidity level can be, for example, about 70%, about 71%,about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%,about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about90.5%, about 91%, about 91.5%, about 92%, about 92.5%, about 93%, about93.5%, about 94%, about 94.5%, about 95%, about 95.5%, about 96%, about96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about99.5%, or about 99.9%.

The CO2 levels in the cell culture incubator can be, for example, about10%, about 9.5%, about 9%, about 8.5%, about 8%, about 7.5%, about 7%,about 6.9%, about 6.8%, about 6.7%, about 6.6%, about 6.5%, about 6.4%,about 6.3%, about 6.2%, about 6.1%, about 6%, about 5.9%, about 5.8%,about 5.7%, about 5.6%, about 5.5%, about 5.4%, about 5.3%, about 5.2%,about 5.1%, about 5%, about 4.9%, about 4.8%, about 4.7%, about 4.6%,about 4.5%, about 4.4%, about 4.3%, about 4.2%, about 4.1%, about 4%,about 3.9%, about 3.8%, about 3.7%, about 3.6%, about 3.5%, about 3.4%,about 3.3%, about 3.2%, about 3.1%, about 3%, about 2.9%, about 2.8%,about 2.7%, about 2.6%, about 2.5%, about 2.4%, about 2.3%, about 2.2%,about 2.1%, about 2%, about 1.9%, about 1.8%, about 1.7%, about 1.6%,about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1%,about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%,about 0.3%, about 0.2%, or about 0.1%.

The cell culture incubator can be used in combination with the culturingconditions described herein, for example, to isolate specific cellpopulations, to induce changes in cells, to introduce exogenousmaterials into cells, to determine biomarker expression, and as adiagnostic tool for patients.

The methods of the invention can be used to increase, for example,transfection and transduction efficiency in cells. Transduction can beused, for example, to introduce a viral vector in a cell. Viral nucleicacid delivery systems can use recombinant viruses to deliver nucleicacids for gene therapy. Non-limiting examples of viruses that can beused to deliver nucleic acids include retrovirus, adenovirus, herpessimplex virus, adeno-associated virus, vesicular stomatitis virus,reovirus, vaccinia, pox virus, and measles virus.

Transfection methods that can be used with methods of the inventioninclude, for example, lipofection, electroporation, calcium phosphatetransfection, chemical transfection, polymer transfection, gene gun,magnetofection, or sonoporation. The transfection can be a stable ortransient transfection. The transfection can be used to transfect DNAplasmids, RNA, siRNA, shRNA, or any nucleic acid. The plasmids canencode, for example, green fluorescent protein (GFP), selectablemarkers, and other proteins of interest. The selectable markers canprovide resistance to, for example, G418, hygromycin B, puromycin, andblasticidin. The transfection method used with a method of the inventioncan further introduce a vector system encoding the CRISPR-Cas9 systeminto a cell.

A method of the invention can increase the transfection or transductionefficiency by, for example, about 2-fold, about 3-fold, about 4-fold,about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold,about 10-fold, about 12-fold, about 14-fold, about 16-fold, about18-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold,about 40-fold, about 45-fold, about 50-fold, about 60-fold, about70-fold, about 80-fold, about 90-fold, or about 100-fold.

Therapeutic Uses

Subjects can be, for example, elderly adults, adults, adolescents,pre-adolescents, children, toddlers, infants. Subjects can be non-humananimals, for example, a subject can be a mouse, rat, cow, horse, donkey,pig, sheep, dog, cat, or goat. A subject can be a patient.

A method of the invention can be used to treat or diagnose, for example,cancer in a subject. A method of the invention can be used to identify atherapeutic, a biomarker, a genetic mutation, an epigenetic marker, or atherapeutic target for cancer. A method of the invention can also beused to develop a library or database of genetic mutations found incancer. A method of the invention can be used for personalized medicine.A method of the invention can be used to determine the effect of atherapeutic on a specific cell type.

A method of the invention can be used, for example, to enrich specificpopulations of cells or induce expression of specific genes, forexample, biomarkers or epigenetic markers. A method of the invention canbe used, for example, to affect the potency of stem cells or somaticcells. For example, a method of the invention can be used to test theability of stem cells to go from, for example, totipotent to, forexample, pluripotent, oligopotent, or unipotent.

The change in gene expression can affect, for example, cell quantity,cell morphology, cell growth, cell motility, cell invasion, or celladhesion.

Genomic, proteomic, and metabolic analysis can be conducted on thecultured cells to, for example, identify biomarkers that can be used fordevelopment of cancer therapies, drug development, cancer vaccines,cancer screening, diagnostics, personalized antibody development,hematopoietic stem cell transplantation, organ transplantation, orcardiovascular disease treatment.

Non-limiting examples of cancers that can be analyzed in a method of theinvention include: acute lymphoblastic leukemia, acute myeloid leukemia,adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma,anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bileduct cancer, bladder cancer, bone cancers, brain tumors, such ascerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas, Burkitt lymphoma, carcinoma of unknown primary origin, centralnervous system lymphoma, cerebellar astrocytoma, cervical cancer,childhood cancers, chronic lymphocytic leukemia, chronic myelogenousleukemia, chronic myeloproliferative disorders, colon cancer, cutaneousT-cell lymphoma, desmoplastic small round cell tumor, endometrialcancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ celltumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoidtumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia,head and neck cancer, heart cancer, hepatocellular (liver) cancer,Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, isletcell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip andoral cavity cancer, liposarcoma, liver cancer, lung cancers, such asnon-small cell and small cell lung cancer, lymphomas, leukemias,macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma,medulloblastoma, melanomas, mesothelioma, metastatic squamous neckcancer with occult primary, mouth cancer, multiple endocrine neoplasiasyndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity andparanasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma,non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer,oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma ofbone, ovarian cancer, ovarian epithelial cancer, ovarian germ celltumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinusand nasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pineal astrocytoma, pineal germinoma,pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia,primary central nervous system lymphoma, prostate cancer, rectal cancer,renal cell carcinoma, renal pelvis and ureter transitional cell cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skincancers, skin carcinoma merkel cell, small intestine cancer, soft tissuesarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma,throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastictumor (gestational), cancers of unknown primary site, urethral cancer,uterine sarcoma, vaginal cancer, vulvar cancer, Waldenströmmacroglobulinemia, and Wilms tumor.

Cell surface marker molecules, such as EPCAM, CD133, EGFR, HER2, or CD20can be used to identify a population of cells. In some embodiments, acombination of cell surface markers, or a cell surface marker signature,can be used to identify a population of cells. In some embodiments, acell surface marker, and/or a cell surface marker signature can be usedin medical diagnosis.

Epigenetic markers that can be assessed using a method of the inventioninclude, for example, DNA methylation, cytosine methylation,hydroxymethylation, histone methylation, lysine acetylation, lysinemethylation, arginine methylation, serine phosphorylation, threoninephosphorylation, protein phosphorylation, protein ubiquitination,protein sumoylation, presence of 5-methylcytosine, histone H3acetylation, or histone H4 acetylation.

Methods that can be used to determine the presence of biological markersinclude, for example, qPCR, RT-PCR, immunofluorescence,immunohistochemistry, western blotting, high-throughput sequencing,ELISA, or mRNA sequencing.

Target cell subpopulations can be used for personalized medicine. Forexample, CTCs and CSCs can be used for chemosensitivity testing wherebychemotherapy regimens can be tested on cultured CTCs. An assessment ofthe effects of chemotherapy drugs on CTCs including, for example, cellviability and cell division, can be done to determine the efficacy of agiven drug.

The methods of the present invention can be used to monitor subjectresponse to a given cancer therapy conducted by serial monitoring of thesubject's CTC population as treatment progresses. Blood samples can beanalyzed on a regular basis, before, during, and after treatment toassess CTC viability.

The methods of the invention can be used to monitor subjects who arecurrently in remission to investigate the potential of cancer relapse.Serial testing of subject blood for CTCs can be conducted on a regularbasis to determine the potential or likelihood for cancer relapse. Insome cases, serial testing can result in earlier detection of relapse.Serial testing can also be used for long-term longitudinal studies.

A method of the invention can be used to collect data about patients forpatient stratification during clinical trials. For example, the presenceof a specific biomarker found in a patient's CTCs can be used to placethe patient in appropriate clinical trial groups, or can be used asexclusion criteria for other clinical trial groups.

The invention described herein can provide data that can be used for amedical professional to treat a patient. Treatment of a patient caninclude diagnosis, prognosis or theranosis. Diagnoses can comprisedetermining the condition of a patient. Diagnosis can be conducted atone time point or on an ongoing basis. For example, a patient can bediagnosed with cancer. In another example, a cancer patient who is inremission can be routinely screened to determine if a cancer relapse hasoccurred. Prognosis can comprise determining the outcome of a patient'sdisease, the chance of recovery, or how the disease will progress. Forexample, identifying CTCs of a certain type can provide information uponwhich a prognosis can be based. Theranosis can comprise determining atherapy treatment. For example, a patient's cancer therapy treatment caninclude chemotherapy, radiation, drug treatment, no treatment, or anycombination thereof. A patient can be monitored, for example by serialblood testing, to measure CTC populations before, during and after apatient undergoes treatment. A positive response to therapy can resultin a decreased CTC viability and lower division rates.

Computer Systems

A method of the invention can be used to, for example, sequence, image,or characterize the collected target cell subpopulations. Furthermethods can be found in PCT/US14/13048, the entirety of which isincorporated herein by reference.

The invention provides a computer system that is configured to implementthe methods of the disclosure. The system can include a computer server(“server”) that is programmed to implement the methods described herein.FIG. 4 depicts a system 400 adapted to enable a user to detect, analyze,and process images of cells and sequence cells. The system 400 includesa central computer server 401 that is programmed to implement exemplarymethods described herein. The server 401 includes a central processingunit (CPU, also “processor”) 405 which can be a single core processor, amulti core processor, or plurality of processors for parallelprocessing. The server 401 also includes memory 410 (e.g. random accessmemory, read-only memory, flash memory); electronic storage unit 415(e.g. hard disk); communications interface 420 (e.g. network adaptor)for communicating with one or more other systems; and peripheral devices425 which may include cache, other memory, data storage, and/orelectronic display adaptors. The memory 410, storage unit 415, interface420, and peripheral devices 425 are in communication with the processor405 through a communications bus (solid lines), such as a motherboard.The storage unit 415 can be a data storage unit for storing data. Theserver 401 is operatively coupled to a computer network (“network”) 430with the aid of the communications interface 420. The network 430 can bethe Internet, an intranet and/or an extranet, an intranet and/orextranet that is in communication with the Internet, a telecommunicationor data network. The network 430 in some cases, with the aid of theserver 401, can implement a peer-to-peer network, which may enabledevices coupled to the server 401 to behave as a client or a server. Themicroscope and micromanipulator can be peripheral devices 425 or remotecomputer systems 440.

The storage unit 415 can store files, such as individual images, timelapse images, data about individual cells, cell colonies, or any aspectof data associated with the invention. The data storage unit 415 may becoupled with data relating to locations of cells in a virtual grid.

The server can communicate with one or more remote computer systemsthrough the network 430. The one or more remote computer systems may be,for example, personal computers, laptops, tablets, telephones, smartphones, or personal digital assistants.

In some situations the system 400 includes a single server 401. In othersituations, the system includes multiple servers in communication withone another through an intranet, extranet and/or the Internet.

The server 401 can be adapted to store cell profile information, suchas, for example, cell size, morphology, shape, migratory ability,proliferative capacity, kinetic properties, and/or other information ofpotential relevance. Such information can be stored on the storage unit415 or the server 401 and such data can be transmitted through anetwork.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) computer readable medium (or software) stored on anelectronic storage location of the server 401, such as, for example, onthe memory 410, or electronic storage unit 415. During use, the code canbe executed by the processor 405. In some cases, the code can beretrieved from the storage unit 415 and stored on the memory 410 forready access by the processor 405. In some situations, the electronicstorage unit 415 can be precluded, and machine-executable instructionsare stored on memory 410. Alternatively, the code can be executed on asecond computer system 440.

Aspects of the systems and methods provided herein, such as the server401, can be embodied in programming. Various aspects of the technologymay be thought of as “products” or “articles of manufacture” typicallyin the form of machine (or processor) executable code and/or associateddata that is carried on or embodied in a type of machine readable medium(e.g., computer readable medium). Machine-executable code can be storedon an electronic storage unit, such memory (e.g., read-only memory,random-access memory, flash memory) or a hard disk. “Storage” type mediacan include any or all of the tangible memory of the computers,processors or the like, or associated modules thereof, such as varioussemiconductor memories, tape drives, disk drives and the like, which mayprovide non-transitory storage at any time for the software programming.All or portions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical, and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless likes, opticallinks, or the like, also may be considered as media bearing thesoftware. As used herein, unless restricted to non-transitory, tangible“storage” media, terms such as computer or machine “readable medium”refer to any medium that participates in providing instructions to aprocessor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, tangible storage medium,a carrier wave medium, or physical transmission medium. Non-volatilestorage media can include, for example, optical or magnetic disks, suchas any of the storage devices in any computer(s) or the like, such maybe used to implement the system. Tangible transmission media caninclude: coaxial cables, copper wires, and fiber optics (including thewires that comprise a bus within a computer system). Carrier-wavetransmission media may take the form of electric or electromagneticsignals, or acoustic or light waves such as those generated during radiofrequency (RF) and infrared (IR) data communications. Common forms ofcomputer-readable media therefore include, for example: a floppy disk, aflexible disk, hard disk, magnetic tape, any other magnetic medium, aCD-ROM, DVD, DVD-ROM, any other optical medium, punch cards, paper tame,any other physical storage medium with patterns of holes, a RAM, a ROM,a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, acarrier wave transporting data or instructions, cables, or linkstransporting such carrier wave, or any other medium from which acomputer may read programming code and/or data. Many of these forms ofcomputer readable media may be involved in carrying one or moresequences of one or more instructions to a processor for execution.

Gas Flow Regulation System

Embodiments of the cell culture incubator include sensors foratmospheric parameters such as oxygen, carbon dioxide, total gaspressure, temperature, and dew point, each of which deliver sensory datato the control unit. Within the control unit, these data are receivedand acted upon by various atmospheric control modules such as an oxygenmodule, a pressure module, a carbon dioxide module, a temperaturemodule, and a dew point module. By way of various control pathways,these modules each engage features and mechanics of a cell cultureincubator gas flow system that operate to establish the atmosphericparameters within the incubators, such as individual gas sources, flowlines, flow valves, pumps, and vents.

FIGS. 24A-24B depict embodiments of a gas flow system 2400 for a cellculture incubator that control the flow of various gases into enclosedenvironment chamber 2401 in order to regulate aspects of the gaseous oratmospheric environment within the chamber. These parametersparticularly include the total gas pressure and the oxygen concentrationof the total gas composition, but further may include carbon dioxide,nitrogen, and water vapor. FIG. 24A is a schematic diagram of anembodiment of gas flow control system 2400 to be used with a cellculture incubator and a method of the invention. FIG. 24B is schematicdiagram of an embodiment of a nitrogen flow system 2450 (which may beconsidered a subsystem of gas flow control system 2400) to be used witha cell culture incubator and a method of the invention. Embodiments ofthe invention, per systems as depicted in FIGS. 24A-24B, are directedtoward creating cell culture conditions that include both low oxygen andhigh pressure, both parameters being regulated independently of eachother.

FIGS. 24A-24B show major components of system 2400 and 2450,respectively, which include an incubator chamber 2401, a chamber door2416 (FIG. 24B) an incubator chamber heater 2402, an air injection pump2403, a recirculation pump 2404, a display 2405, and a control unit2406. Label 2401 refers to a cell culture incubator's enclosedenvironmental chamber, but, for simplicity, may also refer moregenerally to an incubator as a whole. Both pumps 2403 and 2404 are inoperative communication with the interior of enclosed incubator chamber2401. As described earlier in the disclosure, embodiments of the cellculture incubator and its control systems are typically controlled byway of user input via a user interface (FIG. 15), and by automaticaction of pumps and valves by way of sensory feedback from within theincubator chamber 2401, as mediated by control unit 2406 and itscomponent control modules, as described further below.

Gas flow into and out of enclosed environmental chamber 2401 by way ofgas control system 2400 includes controlled input of nitrogen gas 2434,controlled input of carbon dioxide 2432, and controlled input of air2436 (by way of injection pump 2403). The internal atmosphere within theincubator also has an influx and efflux by way of recirculation pump2404, which facilitates a homogeneous mixing of gases within enclosedenvironmental chamber 2401. Influx of nitrogen, carbon dioxide, and air,by way of their respective pumps, is controlled by way of control unit2406, by way of sensors and gas control modules, as described below.

A number of types of sensors may be included within incubator chamber2401 that are responsive to various atmospheric conditions, and whichtransmit sensed data to control unit 2406 and its various controlmodules. These sensors include an oxygen sensor 2410, a carbon dioxidesensor 2411, a pressure sensor 2412, a temperature probe 2413, a dewpoint sensor 2414. In some embodiments of system 2400, one or morepressure sensors may be included that are disposed and configured tomeasure external ambient atmospheric pressure. Various types of oxygensensors are commercially available and suitable for embodiments of theinvention. For example, AMI (Huntington Beach, Calif.) manufacturers anoxygen probe that delivers oxygen level data in terms of concentration.Instruments that deliver concentration data typically make use of areference gas or reference to ambient air. Another example of a suitableoxygen probe is provided by SST (Coatbridge, UK), which delivers oxygenlevel data in terms of partial pressure. Control unit 2406 (via display2405) provides oxygen level data in terms of concentration, even ifsensor data reports oxygen partial pressure. Most fundamentally, thebasic oxygen parameter is its partial pressure, which can be expressedeither directly or by conversion, or by comparison to reference data, asa relative percent of a total atmospheric composition.

In a typical embodiment, data from dew point sensor is directed bycontrol unit 2406 to display 2405, and in some embodiments, control ofhumidity is passive (as for example when humidity is maintained by wayof evaporation of liquid water in the enclosed chamber) without theintervention of a dedicated control module.

A contact sensor 2475 (FIG. 24B) may also disposed at a site interfacingbetween chamber door 2416 that is sensitive to contact between the doorand its frame. In one embodiment, for example, contact sensor 2475 is adepressable button that operates valve 2474. The role of valve 2474 incontrolling gas input, particularly nitrogen input, into incubatorchamber 2401 is described further below.

Within control unit 2406 are several control modules; these include anoxygen module 2420, a carbon dioxide module 2422, a pressure module2424, and a temperature module 2426. Each of these modules receivesensory input from their corresponding sensors, i.e., oxygen sensor2410, carbon dioxide sensor 2411, pressure sensor 2412, and temperatureprobe 2413, respectively. Signals from each of these types of sensor arereceived by corresponding modules and used to formulate instructionsthat are sent to the various gas flow control mechanisms and pumps, asdescribed herein, to achieve the instructed atmospheric parameters.Sensor values, or an algorithm-derived expression thereof, may also beshown in display 2405, as exemplified by FIGS. 16-18.

Control unit 2406 effects control of the atmospheric environment withinenclosed environmental chamber 2401 by several control paths. Displaycontrol path 2441 informs the read out on display 2405. Heater path 2443is responsive to temperature module 2426, and controls the operation ofheater 2402. Recirculation pump 2404 operates at a constant rate thatcan be set, but is typically not subject to sensory feedback control.

Gas control path 2442 is shown in a simplified depiction as a singleline, but it represents control paths for the operation of nitrogen 2434inflow, carbon dioxide 2432 inflow, vent efflux 2438 control, and air2436 injection by way of air injection pump 2403. The control ofnitrogen inflow, carbon dioxide inflow, gas efflux through the vent, andair inflow are all controlled more particularly by valves or flowregulators that are not shown for the sake of simplicity. Nitrogen 2434inflow control is responsive to oxygen module 2420 and pressure module2424. Injection of nitrogen may be used both to increase pressure withinenclosed environmental chamber 2401 and to decrease the oxygenconcentration, as described further below.

In typical operation, cell culture incubator 2401 operates at an oxygenlevel that ranges from an ambient oxygen level to a lower oxygen level,as described earlier. Although some embodiments of incubator 2401 may beconfigured to operate at oxygen levels higher than ambient levels,typical embodiments of incubator do not. A typical operational task,therefore, is to decrease oxygen concentration to a level less than thatof the ambient condition. This oxygen-lowering task is accomplishedprimarily by injection of nitrogen 2434, which is controlled by controlunit 2406 with input from the oxygen module 2420. If the oxygen leveldrifts to level higher than a targeted or instructed level, nitrogeninjected into enclosed environmental chamber 2401 mixes with existinggas composition and drives the oxygen level by dilution. Once theinstructed oxygen level is achieved, control unit 2406 shuts offnitrogen injection.

Referring now particularly to FIG. 24B, and returning to a descriptionof a gas driven safety lock mechanism 2472. For orientation, it can beseen that as a gas such as nitrogen 2434 enters the incubator, it splitsinto two paths: a chamber gas flow path 2460 and a door lock controlpath 2470. Chamber gas flow path 2460 includes intervention of regulator2462 and valve 2464. Details of the control of chamber gas flow path arecovered above in the description of gas control system 2400 as a whole,and as shown in FIG. 24A.

The operation of a cell culture incubator at a higher-than-ambient totalgas pressure is benefited from a construction that is fortified againstgas leakage, and which allows a door to the incubator to opened safely,without undue disturbance of the atmosphere within the incubator, unduedisruption of gas regulation controls, and without unnecessary loss ofgas that being injected in to enclosed environmental chamber 2401.Accordingly, some embodiments of the cell culture incubator include adoor 2416 to the enclosed environmental chamber and a safety lock 2472configured to prevent opening of the door when the enclosedenvironmental chamber is in a pressurized condition.

Some of these the safety lock embodiments 2472 include a pistonconfigured to be able to assume a locked position and an unlockedposition, the locked position of the safety lock being secured by a gaspressure behind the piston, and the unlocked position being assumed by arelease of such gas pressure. In some of these embodiments, the gaspressure is provided by nitrogen, the nitrogen being delivered to apiston chamber behind the piston, the nitrogen being provided to thechamber by way of a piston gas flow control path 2470 from the nitrogensource. Use of nitrogen for the purpose of driving door control path2470 is a practical choice; but oxygen or carbon dioxide, or any othergas being put into the system would also work. Nitrogen is practical forthis use because it is already used in relatively high volume andbecause release of nitrogen into the atmosphere is benign.

Tracking the elements of door lock control path 2470, it can be seenthat nitrogen 2434 encounters locking piston 2472 which is configured topenetrate into enclosed environmental chamber door 2416 in its onposition, the on position being secured by nitrogen pressure behindlocking piston 2472. A contact sensor 2475 (as noted above in thecontext of enumerating the various sensors in the system) is positionedat a point of contact between chamber door 2416 and its enclosing frame,such that it senses whether the door is open or closed, and communicatesthe open/closed status to controller 2406.

During operation of enclosed environmental chamber 2401, door 2416 isclosed and locked by locking piston 2472. An opening sequence beginswith user input to allow the door to open, in response, controller 2406instructs valve 2474 to open, thereby stopping the flow of nitrogen intoa chamber behind the piston, thereby releasing the pressure thatsupports the projection of the piston into door 2416, thereby allowingthe door to be manually opened. In a locking sequence, contact sensor2475 senses that the door has been closed, the controller opens valve2474, thereby resuming the flow of nitrogen into the chamber behind thepiston, thereby driving the front portion of locking piston 2472 intodoor 2416, and securely locking it shut.

Central to the operation of enclosed environment chamber 2401 and to themission of controlling atmospheric or gaseous aspects of the environmentfor the purpose of creating particular effects on cultured cells is theformation of high fidelity and tunable atmosphere typicallycharacterized by low oxygen (lower than the ambient level) and highpressure (higher than the ambient level), these two parameters beingindependently adjustable. It is noteworthy that the goal of instillinghigh pressure, in and of itself, inherently works against instilling lowoxygen. Low oxygen is commonly and reasonably expressed as a percent,as, for example, can be read on display 2405, and as seen in FIGS.16-18. In some embodiments, oxygen sensor 2410 is actually sensing apartial pressure of oxygen gas, an absolute term, not a relative % term.To derive an oxygen percent concentration, control unit 2406 considersboth the partial pressure signal from oxygen sensor 2410 and either atotal gas pressure signal or a reference gas, and by way of a formuladelivers the “oxygen level %” value seen on display 2405.

In addition to oxygen partial pressure being the most basic oxygen levelparameter, partial pressure is also the oxygen parameter of importanceto cells in culture. Total gas pressure, by itself, is also a highlyimportant atmospheric parameter for cells in culture, but it is separatefrom the effects of oxygen, as measured in partial pressure terms. Thisdescription of the oxygen level in terms of a fraction of the total gaspressure is being provided because it makes it clear that increasing thetotal gas pressure also, inherently, increases the partial pressure ofany component gas species within the total gas composition. Accordingly,increasing total gas pressure increases the oxygen partial pressure,which is working against the goal of decreasing oxygen partial pressure.Accordingly, in embodiments of the invention, and to the extent thattotal gas pressure works to counter the goal of creating a low oxygenenvironment, instructions from control unit 2406 to create a low oxygencondition prevail despite the coincident instructions to create a highpressure condition.

EXAMPLES Example 1: Identification of Markers Associated With ProstateCancer

FIG. 5 shows the results of an experiment measuring expression levels ofcancer-associated proteins using samples of CTCs obtained from a sampleof patients with prostate cancer. The CTCs were grown and culturedaccording to a method of the invention to obtain an enriched CTCpopulation. Gene expression of various markers associated with prostatecancer was assessed using qPCR. CD45 expression was assessed viaimmunofluorescence for two subjects, 43-B and 45-C. The white arrowindicates staining for CD45 and the black arrows indicate staining forEPCAM (epithelial cell adhesion molecule)/PSMA. The top panel onlycontains CD45 staining. The markers assessed included androgen receptor(AR), androgen receptor splice variant 7 (AR-V7), EPCAM, prostatic acidphosphatase (PAPS), prostate-specific antigen (KLK3/PSA),prostate-specific membrane antigen (FOLH1/PSMA), v-ets avianerythroblastosis virus E26 oncogene homolog (ERG), prostate cancerantigen 3 (PCA3), Nk3 homeobox 1 (NKX3-1), and chromogranin A orparathyroid secretory protein 1 (CHGA).

The results indicated that PAPS was expressed in all prostate tumorsamples. The expression of other prostate cancer markers differed amongthe samples indicating that CTC colonies can be genetically diversebetween subjects.

Example 2: Identification of Markers Associated With Prostate Cancer

To identify immunotherapeutic targets and stem cell markers that can beexpressed by prostate CTC colonies, 10 to 20 mL of peripheral blood wascollected from over 30 subjects with metastatic CRPC (mCRPC). Eight ofthe subject samples yielded CTC colonies after culturing using a methodof the invention as described in FIGS. 2-3. Four of the samples wereused for qPCR analysis of several markers and were compared to the LNCaP(prostate adenocarcinoma) and PC-3 (prostate adenocarcinoma) cell linesas shows in FIG. 6, which contains the same staining pattern describedfor FIG. 1.

A representative immunofluorescence image is shown with DAPI, WBC,cytokeratin, and PSMA/PSA staining as in FIG. 1. The results indicatedthat several of the patient samples and cell lines expressed theimmunotherapeutic targets programmed death ligand 1 (CD274/PD-L1) andcytotoxic T-lymphocyte-associated protein 4 (CTLA4) and the stem cellmarkers octamer-binding transcription factor 4 (PUSF1/Oct4), SRY (sexdetermining region Y)-box 2 (SOX2), keratin 18, type 1 (KRT18), andkeratin 14, type 1 (KRT14), and that there was upregulation in Oct4,SOX2, and PD-L1.

Example 3: RNA Sequencing of Prostate Cancer CTC Colonies

FIGS. 7-9 display results of a mRNA sequencing analysis to determinedifferential expression of specific signaling pathways among CTCcolonies obtained from a sample of subjects. The CTC colonies wereobtained using a method as described in FIGS. 2-3. The RNA sequence ofthe cells was mapped to specific genes, and the gene counts werenormalized across a collection of samples. Using a non-parametricenrichment algorithm, statistical tests were performed to detectpathways associated with relatively high expression in each sample.False discovery rates were calculated across large collections ofpathways. The enrichment test results were expressed as a falsediscovery rate on the x-axis for each prostate sample RNA profile asseen in FIGS. 7-9. The enrichment for gene expression in differentpathways was different across the samples. Pathways that show enrichmentin prostate CTC colonies included Nerve Growth Factor Receptor Signaling(FIG. 7), Aurora A Signaling (FIG. 8), and Kit Receptor Signaling (FIG.9)

Example 4: EPCAM Expression of Pancreatic CTC Colonies

To determine EPCAM expression in pancreatic CTC colonies, 6 patientswith pancreatic ductal adenocarcinoma (PDAC) were profiled. Apheresedblood samples were collected and cultured to yield CTC colonies. Thecells were stained for cytokeratin 19 (CK19; top left panel, andcircular staining in right panel) and EPCAM (punctate staining aroundcell in bottom left panel, and peripheral staining indicated by whitearrows in right panel) as shown in FIG. 10. Cell binding to thecollagen-based substrate used for culturing of the CTCs led to increasedexpression of EPCAM.

Example 5: Mutations Exhibited by Pancreatic CTC Colonies

Six pancreatic CTC colony samples were analyzed for mutations found inpancreatic cancer as determined from the COSMIC (Catalogue of SomaticMutations in Cancer) database. In the COSMIC database for PDAC tumors,the mutation rate in KRAS is 69%, p53 is 51%, cyclin-dependent kinaseinhibitor 2A (CDKN2A) is 23%, SMAD4 is 21%, AT-rich interactivedomain-containing protein 1A (ARID1A) is 6%, and beta-catenin (CTNNB1)is 2%. For CTC colonies obtained after culturing, 2/6 of the coloniesdisplayed mutations in KRAS, and ⅙ colonies displayed mutations in p53,CDK2NA, and CTNNB1.

Example 6: Gene Expression in Pancreatic and mCRPC CTC Colonies

To determine whether there was differential expression between CTCsobtained from different tumors, PDAC CTCs were compared to mCRPC CTCs.Pancreatic CTCs exhibited increased gene expression in the NANOG, Wnt,insulin-like growth factor 1 (IGFR1), FOXP1, and AR signaling pathways.The RNA sequence of the cells was mapped to specific genes, and the genecounts were normalized across a collection of samples. Using anon-parametric enrichment algorithm, statistical tests were performed todetect pathways associated with relatively high expression in eachsample. False discovery rates were calculated across large collectionsof pathways. The enrichment test results were expressed as a falsediscovery rate on the x-axis for each prostate sample RNA profile asseen in FIGS. 11-12. The enrichment for gene expression in differentpathways was different across the samples. Pathways that show enrichmentin pancreatic CTC colonies over prostate cancer CTCs included the NANOGsignaling pathway (FIG. 11) and the Wnt signaling pathway (FIG. 12).Dendritic cells, lung and T-cells were used as controls, and the rest ofthe samples were CTCs obtained from the subjects.

Example 7: SNP/INDEL Variant Analysis

To determine if CTC fractions displayed more genetic variation thanwhole blood cell controls, single nucleotide polymorphisms (SNPs) andinsertions/deletions (INDELs) were analyzed for six patients with stage4 PDAC. FIG. 13 depicts the results of SNP and INDEL analysis andindicates that both in terms of total events (left panel) and totalnumber of genes with events (right panel), the CTC samples were able touncover more genetic variants compared to whole blood cell controls.

Using a targeted sequencing method, patient samples were assessed for238 genes associated with PDAC. FIG. 14 illustrates the results of thesequencing analysis and shows that CTCs (left side) revealed about 20-30times more SNPs and INDELs compared to whole blood cell controls forspecific genes. The numbers at the top of the chart denote the patientfrom which the sample was obtained.

Example 8: User Interfaces for Displays in a System of the Invention

FIG. 15 depicts a graphical interface that is seen by the user uponinitialization of a system of the invention. In the top-left corner, theuser enters his/her user identification code. In the top-right corner,the user selects the desired option. The quadrant at the bottom-leftcorner indicates the progress of the desired operation. The bottom-rightcorner allows the user to enter his/her password for the system. FIG. 16depicts an additional user interface that can be displayed by a systemof the invention upon logging into the system. The user interface ofFIG. 16 can be configured to be displayed, for example, for a minimumduration of time, until the hardware connectivity is verified, and untilthe user hits the start button.

FIG. 17 is a screen displaying the status of the oxygen level (%),chamber pressure (PSI), temperature (° C.), and carbon dioxide level (%)in the culture chamber. The screen further displays the relativehumidity and experiment time remaining. The user can further enter alarmsettings and time settings using the icons at the bottom of the screen.In this scenario, the oxygen level was 20.0%, the chamber pressure was3.7 PSIG, the temperature was 34.2° C., and the carbon dioxide level was6.3%. The relative humidity was 90%, and the experiment time remainingwas 2 hours 38 minutes and 20 seconds.

FIG. 18 shows that upon selection of a specific parameter, the user cantouch the arrows and decrease or increase the parameter to a desiredvalue. The user can tap the arrow to change the value in smallincrements, or hold down the arrow to change the value at largerincrements. Once the user has reached the desired value, the usertouches the value, and the desired value becomes confirmed.

Example 9: Coating Process for Cell Adhesion

To prepare a surface for covalent binding of cellular proteins, glassslides were prepared by incubating the slides with 0.1 M hydrochloricacid (HCl) for two hours to overnight at room temperature. Then, theglass slides were incubated with 0.1 M NaOH for two hours to overnightat room temperature. The slides were then incubated with 0.5-5%(3-aminopropyl)-trimethoxysilane for two hours to overnight at roomtemperature. Then, the slides were incubated with 0.5-5% glutaraldehyde(diluted in PBS) for 2 hours to overnight at room temperature. The glassslides were then rinsed with water overnight, sterilized under UV lightfor one hour, and then stored dry at room temperature.

The slides were then further treated to facilitate cell binding using amixture containing extracellular matrix (ECM) proteins. The ECM mixcontained from about 0.1 to about 3 mg/mL collagen, from about 0.1 toabout 10 μg/mL fibronectin, and from about 0.1 to about 10 μg/mL of abasement membrane cocktail. The ECM mix was diluted with either aglycine buffer at pH 10 or a DMEM buffer at pH 7 depending on thecellular application.

Example 10: Transfection Efficiency Using a Method of the Invention

DU145 (human prostate cancer) cells were transfected with a GFP plasmidusing electroporation. 5×106 cells/mL were electroporated using aprotocol of 1260 V for 20 ms twice with 50 ng DNA plasmid/μt of the cellresuspension. After transfection, the cells were split into separate 35mm cell culture plates. One plate was placed in a standard CO2incubator, and the other plate was place in an incubator of theinvention. The second plate's incubator was set to 1% O2 and 2 PSIG.After 48 hours, the cells were fixed and imaged for GFP expression usinga fluorescent microscope. The data shown in FIG. 23 indicate that cellsincubated at low oxygen and high pressure showed higher transfectionefficiency (lower panel) than those cells incubated at standardconditions (upper panel) as depicted by a greater number of brightcells.

EMBODIMENTS

The following non-limiting embodiments provide illustrative examples ofthe invention, but do not limit the scope of the invention.

Embodiment 1

A cell culture incubator, wherein the cell culture incubator comprises:a) an enclosed environmental chamber; and b) a control unit, wherein thecontrol unit is operably linked to the enclosed environmental chamber,wherein the control unit comprises a computer program product comprisinga computer-readable medium having computer-executable code encodedtherein, the computer-executable code adapted to encode: (i) an oxygenlevel module, wherein the oxygen level module is encoded to regulate anoxygen level of the enclosed environmental chamber, wherein the oxygenlevel module is encoded to control the removal of oxygen in the enclosedenvironmental chamber to generate a hypoxic oxygen level within theenclosed environmental chamber; (ii) a pressure module, wherein thepressure module is encoded to regulate a pressure of the enclosedenvironmental chamber, wherein the pressure module controls the additionof gas to generate a positive pressure condition in the enclosedenvironmental chamber; (iii) a temperature module, wherein thetemperature module is encoded to regulate a temperature of the enclosedenvironmental chamber; and (iv) a humidity module, wherein the humiditymodule is encoded to regulate a humidity of the enclosed environmentalchamber, wherein each of the oxygen level, pressure, temperature, andhumidity mimics an in vivo microenvironment for a cell, wherein the cellculture incubator reaches each of an instructed oxygen level, pressure,temperature, and humidity within about 20 minutes of receiving an inputof each of the instructed oxygen level, pressure, temperature, andhumidity.

Embodiment 2

The cell culture incubator of embodiment 1, wherein the in vivomicroenvironment is a tumor microenvironment.

Embodiment 3

The cell culture incubator of any one of embodiments 1-2, wherein thecell is a stem cell.

Embodiment 4

The cell culture incubator of any one of embodiments 1-2, wherein thecell is a cancer cell.

Embodiment 5

The cell culture incubator of any one of embodiments 1-2, wherein thecell is a circulating tumor cell.

Embodiment 6

The cell culture incubator of any one of embodiments 1-2, wherein thecell is an immune cell.

Embodiment 7

The cell culture incubator of any one of embodiments 1-6, wherein thecell is obtained from a biological sample.

Embodiment 8

The cell culture incubator of embodiment 7, wherein the biologicalsample is blood.

Embodiment 9

The cell culture incubator of embodiment 7, wherein the biologicalsample is a tumor.

Embodiment 10

The cell culture incubator of embodiment 7, wherein the biologicalsample is saliva.

Embodiment 11

The cell culture incubator of embodiment 7, wherein the biologicalsample is a tissue.

Embodiment 12

The cell culture incubator of any one of embodiments 1-11, wherein thecontrol unit is user-controlled.

Embodiment 13

The cell culture incubator of any one of embodiments 1-11, wherein thecontrol unit is automated.

Embodiment 14

The cell culture incubator of any one of embodiments 1-13, wherein theoxygen module is encoded to maintain a hypoxic oxygen level.

Embodiment 15

The cell culture incubator of any one of embodiments 1-14, wherein thepressure module is encoded to maintain a positive pressure condition.

Embodiment 16

The cell culture incubator of any one of embodiments 1-15, wherein theoxygen level module is encoded to maintain the oxygen level in theenclosed environmental chamber at about 0.1% to about 21%.

Embodiment 17

The cell culture incubator of any one of embodiments 1-16, wherein theoxygen level module is encoded to maintain the oxygen level in theenclosed environmental chamber at about 2%.

Embodiment 18

The cell culture incubator of any one of embodiments 1-17, wherein theoxygen level module is encoded to maintain the oxygen level in theenclosed environmental chamber at about 0.1%.

Embodiment 19

The cell culture incubator of any one of embodiments 1-18, wherein thepressure module is encoded to maintain the pressure in the enclosedenvironmental chamber at from about 1 PSIG to about 5 PSIG.

Embodiment 20

The cell culture incubator of any one of embodiments 1-19, wherein thehumidity module is encoded to maintain the humidity in the enclosedenvironmental chamber at about 85%.

Embodiment 21

The cell culture incubator of any one of embodiments 1-20, wherein thecontrol unit further comprises a computer program product comprising acomputer-readable medium having computer-executable code encodedtherein, the computer-executable code adapted to encode a carbon dioxidemodule, wherein the carbon dioxide module is encoded to regulate acarbon dioxide level of the enclosed environmental chamber.

Embodiment 22

The cell culture incubator of any one of embodiments 1-21, wherein thecell culture incubator further comprises a gas inlet controlled by theoxygen level module.

Embodiment 23

The cell culture incubator of any one of embodiments 1-22, wherein thecell culture incubator further comprises a gas inlet controlled by thepressure module.

Embodiment 24

The cell culture incubator of any one of embodiments 1-23, wherein thecell culture incubator further comprises a water humidity traycontrolled by the humidity module.

Embodiment 25

The cell culture incubator of any one of embodiments 1-24, wherein thecell culture incubator further comprises a heating element controlled bythe temperature module.

Embodiment 26

The cell culture incubator of any one of embodiments 1-25, wherein thecell culture incubator is configured to accept a cell culture plate.

Any one or more features of any embodiment of the inventions disclosedherein may be combined with any one or more other features of any otherembodiment of the inventions, without departing from the scope of theinvention. It should also be understood that while some theoreticalconsiderations may have been provided to further an understanding ofembodiments of the invention, the claims to the invention are not boundby such theory. It should also be understood that the invention is notlimited to the embodiments that are described or depicted herein forpurposes of exemplification, but is to be defined only by a fair readingof claims appended to the patent application, including the full rangeof equivalency to which each element thereof is entitled.

1-26. (canceled)
 27. A cell culture incubator comprising: an enclosedenvironmental chamber; and a control unit operably linked to theenclosed environmental chamber, wherein the control unit comprises anoxygen module and a pressure module, wherein the control unit isconfigured to regulate each of an oxygen level and a total gas pressurewithin the enclosed environmental chamber, and wherein the control unitis adapted to: a) provide instructions to the oxygen module to regulatethe oxygen level to an instructed hypoxic oxygen level; and b) provideinstructions to the pressure module to regulate the total gas pressureto an instructed positive pressure level, wherein the regulation of theoxygen level to the instructed hypoxic level prevails despite an oxygenpartial pressure increasing effect of a positive pressure condition, perthe instructed positive pressure level.
 28. The cell culture incubatorof claim 27, further comprising: an oxygen sensor configured to measurethe oxygen level within the enclosed environmental chamber and to conveyan informative signal to the oxygen module; and a pressure sensorconfigured to measure the total atmospheric gas pressure within theenclosed environmental chamber and to convey an informative signal tothe pressure module.
 29. The cell culture incubator of claim 27, furthercomprising a nitrogen source operably connected to the enclosedenvironmental chamber, wherein a flow of nitrogen is regulated by thecontrol unit.
 30. The cell culture incubator of claim 29, wherein theregulated nitrogen flow is directed into the enclosed environmentalchamber by way of a chamber gas flow path, and wherein the regulatednitrogen flow comprises a response to oxygen sensor data via the oxygenmodule, wherein in the response to a sensed oxygen level that is abovethe instructed oxygen level, the response comprises an instruction toflow nitrogen into the environmental chamber.
 31. The cell cultureincubator of claim 30, wherein as a result of a dilution of oxygenwithin the enclosed environmental chamber by the flow of nitrogen, thesensed oxygen level reaches the instructed oxygen level, and wherein thecontrol unit then instructs a cessation of the nitrogen flow into theenvironmental chamber.
 32. The cell culture incubator of claim 29,wherein the regulated nitrogen flow is directed into the enclosedenvironmental chamber by way of a chamber gas flow path, and wherein theregulated nitrogen flow comprises a response to pressure sensor data byway of the pressure module, wherein the response to a pressure levelthat is below the instructed pressure level comprises an instruction toflow nitrogen into the environmental chamber.
 33. The cell cultureincubator of claim 32, wherein as a result of an increase in pressurelevel within the enclosed environmental chamber by the flow of nitrogen,the pressure level comes reaches the instructed pressure level, andwherein the control unit then instructs a cessation of the nitrogen flowinto the environmental chamber.
 34. The cell culture incubator of claim28, wherein the regulation of pressure within the enclosed environmentalchamber by the control unit comprises a response to the pressure sensor,as mediated by the pressure module.
 35. The cell culture incubator ofclaim 34, wherein the regulation of pressure within the enclosedenvironmental chamber comprises a response to a pressure lower than theinstructed pressure level, and wherein the response to the high pressurecomprises an inflow of nitrogen.
 36. The cell culture incubator of claim34, wherein the regulation of pressure within the enclosed environmentalchamber comprises a response to a pressure lower than the instructedpressure level, and wherein the enclosed environmental chamber furthercomprises a carbon dioxide source operably connected to the enclosedenvironmental chamber, wherein a flow of carbon dioxide is regulated bythe control unit, and wherein the response to the high pressurecomprises an inflow of carbon dioxide.
 37. The cell culture incubator ofclaim 36, wherein the regulation of pressure within the enclosedenvironmental chamber comprises a response to a pressure lower than theinstructed pressure level, and wherein the response to the low pressurecomprises a cessation of inflow of carbon dioxide or inflow of nitrogen.38. The cell culture incubator of claim 36, wherein the regulation ofcarbon dioxide flow into the enclosed environmental chamber by thecontrol unit comprises a response to a carbon dioxide sensor configuredto measure the carbon dioxide level within the enclosed environmentalchamber.
 39. The cell culture incubator of claim 36, wherein theregulation of carbon dioxide flow into the enclosed environmentalchamber by the control unit comprises a response to the pressure sensor,as mediated by the pressure module.
 40. The cell culture incubator ofclaim 27, wherein the oxygen level within the enclosed environmentalchamber is regulated by the oxygen module, and wherein the oxygen moduleprovides instructions to regulate any one or both of a flow of nitrogenor a flow of carbon dioxide into the enclosed environmental chamber. 41.The cell culture incubator of claim 27, wherein the instructions toregulate to an instructed hypoxic level comprise instructions to adjustthe oxygen level to a value within a range of about 0.1% to about 21%oxygen.
 42. The cell culture incubator of claim 27, wherein theinstructions to regulate to an instructed hypoxic level compriseinstructions to adjust the oxygen level to a value within a range ofabout 1.0% to about 12% oxygen.
 43. The cell culture incubator of claim27, wherein the instructions to regulate to an instructed hypoxic levelcomprise instructions to adjust the oxygen level to a value within arange of about 2% to about 6% oxygen.
 44. The cell culture incubator ofclaim 27, wherein the pressure level within the enclosed environmentalchamber is regulated by the pressure module, wherein the pressure moduleprovides instructions to regulate any one or more of a flow of nitrogenor a flow of carbon dioxide.
 45. The cell culture incubator of claim 27,wherein the instructions to regulate pressure to an instructed positivepressure level comprise instructions to adjust the pressure to a valuewithin a range of about 0.5 PSIG to about 30 PSIG.
 46. The cell cultureincubator of claim 27, wherein the instructions to regulate pressure toan instructed positive pressure level comprise instructions to adjustthe pressure to a value within a range of about 1.0 PSIG to about 20PSIG.
 47. The cell culture incubator of claim 27, wherein theinstructions to regulate pressure to an instructed positive pressurelevel comprise instructions to adjust the pressure to a value within arange of about 2.0 PSIG to about 10 PSIG.
 48. The cell culture incubatorof claim 27, wherein the instructions to regulate pressure to aninstructed positive pressure level comprise instructions to adjust thepressure to a value within a range of about 2.5 PSIG to about 5.0 PSIG.